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TGF-beta2 in aqueous humor suppresses S-phase entry in cultured corneal endothelial cells
K H Chen1, D L Harris, N C Joyce
1Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA.
Purpose:
Corneal endothelium in vivo is arrested in G1, the phase of the cell cycle that prepares cells for DNA synthesis. In many cell types, transforming factor (TGF)-beta inhibits proliferation by inducing G1-phase arrest. Evidence indicates that corneal endothelial cells synthesize mRNA for TGF-beta1 and are also bathed in aqueous humor that contains TGF-beta2 (mainly in a latent form). As such, this cytokine may maintain the corneal endothelium in a G1-phase-arrested state in vivo. The purpose of these studies was to determine the effect of exogenous TGF-beta2 and TGF-beta2 in aqueous humor on DNA synthesis in cultured corneal endothelial cells.
Methods:
Rat corneal endothelial cells were grown in explant culture and identified by morphology and reverse transcription-polymerase chain reaction using primers for specific corneal cell markers. Subconfluent cells were synchronized in the G0 phase (quiescence) by serum starvation for 24 hours. Serum was then added to the cells in the presence or absence of exogenous TGF-beta2 or activated rat aqueous humor. [3H]Thymidine was added, and radioactivity was measured at various time points to detect DNA synthesis. Preincubation of exogenous TGF-beta2 or activated rat aqueous humor with neutralizing antibody was used to test for cytokine specificity.
Results:
A linear increase in [3H]thymidine incorporation began approximately 16 hours after serum addition, and peak incorporation occurred at approximately 24 hours. Exposure of cells to serum plus TGF-beta2 suppressed [3H]thymidine incorporation in a dose-dependent manner at concentrations ranging from 5 pg/ml to 5 ng/ml. [3H]Thymidine incorporation was also suppressed in cells exposed to serum plus rat aqueous humor diluted 1:10. Neutralizing antibody reversed the effects of both exogenous TGF-beta2 and aqueous humor.
Conclusions:
Exogenous TGF-beta2 and TGF-beta2 in aqueous humor suppress S-phase entry of rat corneal endothelial cells. These results suggest that this cytokine in aqueous humor could help maintain the corneal endothelium in a G1-phase-arrested state in vivo.
Insights
Transforming growth factor-beta2 (TGF-beta2) in aqueous humor suppresses DNA synthesis in corneal endothelial cells. This suggests TGF-beta2 helps maintain corneal endothelium quiescence in vivo.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Biology
Background:
- Corneal endothelium cells are arrested in the G1 phase of the cell cycle.
- Transforming growth factor-beta (TGF-beta) is known to inhibit cell proliferation by inducing G1-phase arrest.
- Corneal endothelial cells express TGF-beta1 mRNA and are exposed to TGF-beta2 in aqueous humor.
Purpose of the Study:
- To investigate the effect of exogenous TGF-beta2 and TGF-beta2 present in aqueous humor on DNA synthesis in cultured corneal endothelial cells.
Main Methods:
- Rat corneal endothelial cells were cultured and synchronized in G0 phase via serum starvation.
- Cells were treated with serum in the presence or absence of exogenous TGF-beta2 or activated rat aqueous humor.
- DNA synthesis was measured by [3H]thymidine incorporation, and cytokine specificity was confirmed using neutralizing antibodies.
Main Results:
- Exogenous TGF-beta2 dose-dependently suppressed [3H]thymidine incorporation.
- Rat aqueous humor also suppressed DNA synthesis in corneal endothelial cells.
- Neutralizing antibodies blocked the inhibitory effects of both exogenous TGF-beta2 and aqueous humor.
Conclusions:
- TGF-beta2, both exogenous and within aqueous humor, inhibits S-phase entry in rat corneal endothelial cells.
- These findings support the role of TGF-beta2 in maintaining the corneal endothelium's G1-phase-arrested state in vivo.
